异种骨支架与人骨髓间充质干细胞相容性的实验研究

发布时间：2018-07-17 06:00

【摘要】：目的：(1)研究脱脂、脱蛋白后猪松质骨支架材料的生物学性能。(2)研究脊柱术中出血中的hBMSCs的细胞学特性。(3)探讨不同方法改性的猪松质骨支架材料的细胞相容性情况。方法：(1)通过超声联合化学方法对猪松质骨支架材料进行脱脂、脱蛋白去抗原处理。(2)大体观察骨支架材料的形貌特征,组织切片HE染色观察材料的组织学结构,比重法检测支架材料的孔隙率,检测支架材料的pH值及微生物生长情况。(3)通过密度梯度离心法分离脊柱手术出血中的hBMSCs;倒置显微镜观察细胞生长情况,流式细胞仪分析第三代hBMSCs表面分子表达情况。对第三代hBMSCs细胞进行成骨诱导培养,在诱导后第3、7、14、21天检测细胞内碱性磷酸酶活性；在诱导培养第21天对矿化结节行茜素红染色。(4)采用不同材料包被支架材料对支架材料改性,分为A、B、C三组,A组为胎牛血清包被,B组为I型胶原蛋白包被,C组为对照组。每块材料上加入浓度为1×109/ml的细胞悬液250μ1。分别在第6、12、24、48小时采用胰蛋白酶消化细胞计数,计算三组的细胞粘附率；在第3、6、9、12、14天采用MTT法观察三组的细胞增殖情况。结果：(1)经过处理的骨支架材料外观呈白色,其表面具有多孔结构微孔壁光滑,HE染色镜下观察到网状骨小梁,骨小梁结构完整,无破坏,骨髓腔中血细胞、脂肪细胞去除,亦未见基质细胞残留,椭圆形骨陷窝空虚,未见骨细胞核及其它结构。骨支架材料浸泡液pH值第1-7天平均7.356±0.034,呈中性。比重法测得骨支架材料的孔隙率高达81.34%士4.31%。在24、48、72小时三个检测时间点,浸泡骨支架材料的完全培养基色泽清亮,无混浊及悬浮物,镜下观察,亦未发现细菌及真菌生长。(2)脊柱外科术中出血中分离出原代hBMSCs贴壁生长,呈梭形、多角形。流式细胞仪分析第三代hBMSCs高表达CD73、CD90、CD105,低表达CD14、CD19、CD34、CD45。 hBMSCs经成骨诱导分化后,随着时间的延长,碱性磷酸酶活性增高。诱导分化培养21天后茜素红染色显示成骨细胞体外矿化特性。(3)采用胰蛋白酶消化法计计算细胞粘附率,hBMSCs粘附率在不同方法处理的支架上细胞粘附时相变化的趋势不同,比较结果细胞粘附率B组A组C组。采用MTT法检测hBMSCs增殖情况,C组细胞增值情况较A、B两组增值情况比较均具有统计学差异(P0.05)；而第3、6、9、12、14天A、B两组之间细胞增殖情况比较无统计学差异(P0.05)。结论：(1)经脊柱术中出血分离的hBMSCs细胞符合间充质细胞特征,可在体外有效培养扩增。(2)经脱脂脱蛋白处理的异种骨支架其孔隙率、pH值均适合细胞生长,经过工型胶原蛋白及胎牛血清包被支架材料,I型胶原蛋白包被支架较胎牛血清更适于hBMSCs粘附,两者均可促进hBMSCs在支架材料上生长。
[Abstract]:Objective: (1) to study the biological properties of porcine cancellous bone scaffolds after degreasing and deproteinizing. (2) to study the cytological characteristics of hBMSCs in intraoperative spinal hemorrhage. (3) to investigate the cytocompatibility of different modified porcine cancellous bone scaffolds. Methods: (1) the porcine cancellous bone scaffold was treated by ultrasonic and chemical methods. (2) the morphology of the scaffold was observed and the histological structure of the scaffold was observed by HE staining. Specific gravity method was used to detect the porosity of scaffolds, pH value of scaffold materials and microbial growth. (3) hBMSCs were separated from spinal bleeding by density gradient centrifugation, and cell growth was observed by inverted microscope. The surface molecular expression of the third generation hBMSCs was analyzed by flow cytometry. The third generation of hBMSCs cells were cultured by osteoblast induction, the activity of alkaline phosphatase was detected on the 3rd day after induction, and the mineralized nodules were stained with alizarin red on the 21st day after induction. (4) the scaffold materials were modified with different materials. Group A was divided into three groups: group A: fetal bovine serum capsule, group B, type I collagen coating, group C, control group. A cell suspension of 1 脳 109/ml was added to each material. The cell adhesion rate of the three groups was calculated by trypsin digestion at 48 hours and the cell proliferation of the three groups was observed by MTT assay on the 3rd day. Results: (1) the appearance of the treated bone scaffold was white, and the surface of the scaffold had a porous structure with a smooth wall. The meshwork was observed under HE staining. The bone trabeculae were intact, without destruction, blood cells and fat cells were removed from the medullary cavity. No stromal cells remained, oval bone lacunae were empty, bone nuclei and other structures were not found. The mean pH value of immersion solution of bone scaffold was 7.356 卤0.034 on day 1-7, which was neutral. The porosity of bone scaffold material measured by specific gravity method was 81.34% 卤4.31%. At three detection time points of 24: 48 ~ 72 hours, the complete culture medium for immersing bone scaffold materials was clear in color, free of turbidity and suspensions, and no bacteria or fungi were found under microscope. (2) Primary hBMSCs were isolated from spinal surgery bleeding to grow on the wall. It is fusiform and polygonal. Flow cytometry was used to analyze the high expression of CD73, CD90, CD105, and the low expression of CD14, CD19, CD34, CD45, CD45. after osteogenic induction, the activity of alkaline phosphatase increased with the prolongation of time. After 21 days of differentiation and culture, alizarin red staining showed the mineralized characteristics of osteoblasts in vitro. (3) the cell adhesion rate of hBMSCs was calculated by trypsin digestion method. Results the cell adhesion rate was compared in group B, group A, group C. MTT assay was used to detect the proliferation of hBMSCs. The proliferation of hBMSCs in group C was significantly higher than that in group A (P0.05), but there was no significant difference between group C and group A (P0.05). Conclusion: (1) the hBMSCs cells isolated by intraoperative spinal hemorrhage accord with the characteristics of mesenchymal cells and can be effectively cultured and amplified in vitro. (2) the porosity and pH value of the xenograft scaffolds treated with degreasing and deproteinization are suitable for cell growth. Type I collagen coated scaffold was more suitable for adhesion of hBMSCs than fetal bovine serum, both of which could promote the growth of hBMSCs on scaffold materials.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R318.08

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